The world's most widely grown fruit crop, the grape (Vitis sp.), is cultivated on all continents except Antarctica. However, major grape production centers are in European countries (including Italy, Spain, and France), which constitute about 70% of the world grape production (Mullins et al., Biology of the Grapevine, Cambridge, U.K.:University Press (1992)). The United States, with 300,000 hectares of grapevines, is the eighth largest grape grower in the world. Although grapes have many uses, a major portion of grape production (.about.80%) is used for wine production. Unlike cereal crops, most of the world's vineyards are planted with traditional grapevine cultivars, which have been perpetuated for centuries by vegetative propagation. Several important grapevine virus and virus-like diseases, such as grapevine leafroll, corky bark, and Rupestris stem pitting ("RSP"), are transmitted and spread through the use of infected vegetatively propagated materials. Thus, propagation of certified, virus-free materials is one of the most important disease control measures. Traditional breeding for disease resistance is difficult due to the highly heterozygous nature and outcrossing behavior of grapevines, and due to polygenic patterns of inheritance. Moreover, introduction of a new cultivar may be prohibited by custom or law. Recent biotechnology developments have made possible the introduction of special traits, such as disease resistance, into an established cultivar without altering its horticultural characteristics.
Many plant pathogens, such as fungi, bacteria, phytoplasmas, viruses, and nematodes can infect grapes, and the resultant diseases can cause substantial losses in production (Pearson et al., Compendium of Grape Diseases, American Phytopathological Society Press (1988)). Among these, viral diseases constitute a major hindrance to profitable growing of grapevines. About 34 viruses have been isolated and characterized from grapevines. The major virus diseases are grouped into: (1) the grapevine degeneration caused by the fanleaf nepovirus, other European nepoviruses, and American nepoviruses, (2) the leafroll complex, and (3) the rugose wood complex (Martelli, ed., Graft Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, FAO, UN, Rome, Italy (1993)).
Rugose wood (RW) complex is a term to describe a group of graft-transmissible diseases which are important and widespread on grapevines grown world-wide. Symptoms of RW are characterized by pitting, grooving, or distortion to the woody cylinder of the grapevine scion, rootstock, or both. Based on symptoms developed on different indicator plants after graft inoculation, RW complex can be divided into four components: Kober 5BB stem grooving (KSG), LN 33 stem grooving (LNSG), grapevine corky bark (GCB), and Rupestris stem pitting (RSP) (Martelli, "Rugose Wood Complex," in Graft-Transmissible Diseases of Grapevines, Handbook for Detection and Diagnosis, pp. 45-54, Martelli, ed., Food and Agriculture Organization of the United Nations, Rome, Italy (1993)). Because RW can cause severe decline and death to grapevines (Savino et al., "Rugose Wood Complex of Grapevine: Can Grafting to Vitis Indicators Discriminate Between Diseases?", in Proceedings of the 9th Meetings of the International Council for the Study of Viruses and Virus Diseases of the Grapevine, Anavim, Israel (1989); Credi and Babini, "Effect of Virus and Virus-like Infections on the Growth of Grapevine Rootstocks," Adv. Hort. Sci., 10:95-98 (1996)), it has been included in healthy grapevine detection schemes used in major grapevine growing countries including Italy, France, and the United States.
RSP was discovered in California in the late 1970s (Prudencio, "M. Sc. Thesis: Comparative Effects of Corky Bark and Rupestris Stem Pitting Diseases on Selected Germplasm Lines of Grapes," University of California, Davis, Calif., 36 pages (1985); Goheen, "Rupestris Stem Pitting," in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988) ("Goheen")). The disease was defined by Goheen as follows: after graft inoculation with a chip bud from an infected grapevine, the woody cylinder of the indicator plant Vitis rupestris Scheele St. George ("St. George") develops a narrow strip of small pits extending from the inoculum bud to the root zone. Grafted St. George plants were checked for wood symptoms 2 to 3 years after inoculation. In contrast to GCB, which elicits pitting and grooving on St. George and LN 33, RSP does not produce symptoms on the latter (Goheen, "Rupestris Stem Pitting," in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988)).
RSP is probably the most common component of the RW complex on grapevines. Surveys in California revealed a high disease incidence in many grapevine cultivars imported from Western Europe and Australia (Goheen, "Rupestris Stem Pitting," in Compendium of Grape Diseases, p. 53, Pearson and Goheen, eds., American Phytopathological Society Press, St. Paul, Minn., USA (1988)). An examination of indexing records in California compiled over 23 years revealed RSP infection in 30.5% of 6482 grapevine selections introduced from around the world (Golino and Butler, "A Preliminary Analysis of Grapevine Indexing Records at Davis, Calif.," in Proceedings of the 10th Meeting of the ICVG, pp. 369-72, Rumbos et al., eds., Volos, Greece (1990)). Indexing in New York State showed that 66% of 257 grapevines tested on St. George developed typical small pits below the inoculum bud or around the woody cylinder (Azzam and Gonsalves, Abstract: "Survey of Grapevine Stem-Pitting in New York and Isolation of dsRNA from a Grapevine Selection Infected with Stem Pitting," Phytopathology 78:1568 (1988)). Furthermore, several reports have indicated that RSP is the most frequently detected component of the RW complex in Italy (Borgo and Bonotto, "Rugose Wood Complex of Grapevine in Northeastern Italy: Occurrence of Rupestris Stem Pitting and Kober Stem Grooving," in Extended Abstracts of the 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine (ICVG), pp. 61-62, Gugerli, ed., Montreux, Switzerland (1993); Credi, "Differential Indexing Trials on Grapevine Rugose Wood Syndrome," Extended Abstracts of the 11th Meeting of the International Council for the Study of Viruses and Virus Diseases of the Grapevine (ICVG), p. 63, Gugerh, P., ed., Montreux, Switzerland (1993)).
The effect of RSP on growth, yield, and grapevine quality is not well understood and, thus, subject to debate. The reason for this ambiguity is the absence of a rapid and sensitive diagnostic tool. RSP is the most difficult grapevine disease to diagnose. Serological or molecular methods are not available for diagnosing RSP. Biological indexing on St. George, as described above, has remained the only approach to diagnose RSP. Biological indexing is labor intensive, time consuming (i.e., often requiring up to about three years to obtain results), and, by its very nature, subjective. Moreover, symptoms on St. George can be variable and not exactly as those defined by Goheen. In particular, Credi, "Characterization of Grapevine Rugose Wood Sources from Italy," Plant Disease, 82:1288-92 (1997), recently showed that some RSP infected grapevines induced pitting that is restricted to below the inoculum bud, while others induced pitting around the woody cylinder of inoculated St. George. Thus, the present method of identifying the presence of RSP is not entirely adequate.
The etiology of RSP is unknown. Efforts to isolate virus particles from RSP-infected grapevines and to mechanically transfer the causal virus(es) to herbaceous host plants failed (Azzam and Gonsalves, "Detection of in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered," Plant Disease, 75:96-964 (1991)). However, a major dsRNA species of ca. 8.3 kb, accompanied by a smaller dsRNA of ca. 7.6 kb, was consistently isolated from one Pinot Gris and four Pinot Noir clones that had been indexed positive for RSP (Walter and Cameron, "Double-Stranded RNA Isolated from Grapevines Affected by Rupestris Stem Pitting Disease," Am. J. of Enology and Viticulture, 42:175-79 (1991)). In addition, a third dsRNA of ca. 5.5 kb was observed in three clones. Likewise, an apparently similar dsRNA species of ca. 8.0 and 6.7 kbp was isolated from dormant canes of RSP-infected grapevines collected from California, Canada, and New York (Azzam and Gonsalves, "Detection of dsRNA in Grapevines Showing Symptoms of Rupestris Stem Pitting Disease and the Variabilities Encountered," Plant Disease, 75:960-64 (1991)). Six of eight Californian and three of five Canadian samples contained these two dsRNA species. However, results of New York samples were not consistent. Among eight RSP infected grapevine selections tested, only one showed these two dsRNAs. Using explants growing in tissue culture as source materials, dsRNA of ca. 359 bp was isolated from 21 of 31 grapevine cultivars, all of which were previously indexed on St. George and considered to be infected with RSP (Monette et al., "Double-Stranded RNA from Rupestris Stem Pitting-Affected Grapevines," Vitis, 28:137-44 (1989)).
In view of the serious risk RSP poses to vineyards and the absence of an effective treatment of it, the need to prevent this affliction continues to exist. Moreover, the absence of a rapid and accurate diagnostic assay prevents proper identification of RSP. The present invention is directed to overcoming these deficiencies in the art.